Rationale: Pulmonary infection in CF is polymicrobial and it is possible that anaerobic bacteria, not detected by routine aerobic culture methods, reside within infected anaerobic airway mucus. Objectives: To determine whether anaerobic bacteria are present in the sputum of CF patients. Methods: Sputum samples were collected from clinically stable adults and bronchoalveolar lavage fluid (BALF) samples from children with CF. Induced sputum samples were collected from healthy volunteers who did not have CF. All samples were processed using anaerobic bacteriological techniques and bacteria within the samples were quantified and identified. Measurements and Main Results: Anaerobic species primarily within the genera Prevotella, Veillonella, Propionibacterium and Actinomyces were isolated in high numbers from 42/66 (64%) sputum samples from adult CF patients. Colonisation with P. aeruginosa significantly increased the likelihood that anaerobic bacteria would be present in the sputum. Similar anaerobic species were identified in BALF from paediatric CF patients. Although anaerobes were detected in induced sputum samples from 16/20 volunteers, they were present in much lower numbers and were generally different species to those detected in CF sputum. Species dependent differences in the susceptibility of the anaerobes to antibiotics with known activity against anaerobes were apparent with all isolates susceptible to meropenem. Conclusions: A range of anaerobic species are present in large numbers in the lungs of CF patients. If these anaerobic bacteria are contributing significantly to infection and inflammation in the CF lung, informed alterations to antibiotic treatment to target anaerobes, in addition to the primary infecting pathogens, may improve management.